fats and oils), there are two suitable methods, the second being the more
(i-) The alcohol method, according to Forster and Riechelmann x : 50
grams of the fat and 75 c.c. of 95% alcohol are boiled for 5-10 minutes in
a flask fitted with a reflux condenser, the alcoholic liquid being separated
while still hot and the boiling repeated with a further 75 c.c. of alcohol.
The united alcoholic liquids, which contain the unsaponifiable substances,
are boiled with 15 c.c. of 30 % sodium hydroxide solution until saponifica-
t ion is complete, the liquid being evaporated almost to dryness and the
residue extracted with ether. The ethereal solution is evaporated to dry-
ness and the residue left taken up in a little ether, filtered and again
evaporated. The final residue is dissolved in a little boiling 95% alcohol
containing a few drops of dilute acetic acid and crystallised.
Repetition of the crystallisation from a little boiling absolute alcohol
yields the sterols (cholesterol and phytosterol) ready for microscopic exami-
nation. With practice and by comparisons of mixtures of known com-
position, the crystalline form observed under the microscope indicates if
the cholesterol is pure or mixed with phytosterol; the characteristic form
of the latter is evident in mixtures1 containing only 5% of vegetable oil (see
figures given under General Methods, 19).
The acetyl compound is then prepared as follows : All the crystals,
together with the last mother-liquor, are freed from alcohol by heating in
a glass dish on a water-bath, the residue being boiled for a moment with
3-5 c.c. of acetic anhydride, the dish being covered with a clock-glass mean-
while ; the cover is then removed and the solution evaporated to dryness
on a water-bath. The residue is dissolved in boiling absolute alcohol (about
20 c.c.) and the solution left to crystallise at the ordinary temperature.
When about two-thirds of the alcohol have evaporated, the crystals are
collected in a small filter and washed with 2-3 c.c. of 95% alcohol, the filter
being then dried by pressing between filter-paper and the crystals redis-
solved in 5-10 c.c. of boiling absolute alcohol and left to crystallise. This
operation is repeated three times. After the third crystallisation, the
melting point of the crystals is determined, two further crystallisations
followed by determinations of the melting point being carried out (see later).
(2) Digitonin methodz: 50 grams of the fat are dissolved in about
120 c.c. of chloroform and the liquid heated on the water-bath at about
60° with 20 c.c. of a i% solution of digitonin in 96% alcohol, with frequent
shaking ; it is then left at rest overnight. The cholesterol and phytosterol
combine with the digitonin forming insoluble products (digitonidas), which
are deposited at the bottom of the chloroform solution of the fat. The
liquid is filtered and the precipitate washed on the filter with chloroform
and dried in the air.
1 Zeitschr, fur offentl. Chem,, 1897, p. 10. *
2 This method may be applied under the conditions laid down by Marcusson and
Schilling (Chem. Zeit., 1913, p. 1001) or by Klostermann, Fritzsche, or Klostermann
and Opitz (Zeit&chr. Unt. Nahr. Genussmittel, 1913, XXVI, pp. 433, 614, and 1914,
XXVII, p. 713). The method now described is deduced from these, with modifications
found valuable as a result of tests made in the Italian Central Customs Laboratory by
Dr. L. Settimj.alcohol in all proportions and in acetic acid in the cold. It is, however,